Abstract Jet-lag, shift-work and disturbances in sleep-activity cycles all contribute to degrade mental and physical well-being. To begin addressing the chronobiological bases for such pathophysiologies, this proposal seeks to describe the neural basis to generate and refine the timing signals that organize and trigger daily rhythmic physiology. Here I outline proposals for three related yet independent studies of circadian neurophysiology. Recent advances in imaging and data analysis can record network phenomena with increasing spatial and temporal precision. The circadian pacemaker system produces physiological activity both spontaneously and rhythmically, which promotes an in-depth analysis. We have introduced planar illumination methods to perform 24 hr in vivo brain-wide scans of the Drosophila circadian neural circuit. That work outlines a new framework for how the circadian network encodes time: a pacemaker network whose internal clocks are strongly synchronized, which nevertheless displays sequential activation by different identified pacemaker groups across the day. Furthermore pacemaker cell interactions, principally in the form of multi-hour neuropeptide-mediated delays, appear to be the preponderant mechanism by which the sequential activities of pacemakers are organized. Therefore, the scientific premise for this project rests on the need to better understand the neural basis for the operations of this timing circuit and its modulation. Here I propose work to further real-time in vivo studies of the brain network that is composed of the core ~150 Drosophila circadian pacemaker neurons. To provide a better understanding of neuronal properties of the pacemaking network, and to extend the scope of our initial studies, we will pursue three Aims. Pacemaker cell interactions are the keys to understanding the dynamic relationships that govern the sequence and tempo of network outputs, and to-date our knowledge is limited to only a few such signals. Thus Aim 1 will systematically test pacemaker cell interactions across the network with chemogenetic control agents, using Ca2+ as a reporter. Aim 2 seeks to extend the scope of our work beyond Ca2+ signals by employing a genetic realtime reporter for cyclic nucleotides, which are established 2nd messengers in the circadian circuit but whose in vivo dynamics are poorly defined. Finally, Aim 3 will study dopamine signaling within the circadian circuit ? we will define spontaneous 24 hr patterns of dopamine cell activity in vivo and test two hypotheses concerning the putative functions of dopamine signals in the pacemaker network. Together these efforts will provide multi-layered information regarding the dynamic state of the pacemaker system network-wide in vivo for the course of the entire day.